The present invention relates to the field of molecular synthesis. More specifically, the invention provides systems and methods for directed synthesis of diverse molecular sequences on substrates.
Methods for preparing different polymers are well known. For example, the xe2x80x9cMerrifieldxe2x80x9d method, described in Atherton et al., xe2x80x9cSolid Phase Peptide Synthesis,xe2x80x9d IRL Press, 1989, which is incorporated herein by reference for all purposes, has been used to synthesize peptides on a solid support. In the Merrifield method, an amino acid is covalently bonded to a support made of an insoluble polymer or other material. Another amino acid with an alpha protecting group is reacted with the covalently bonded amino acid to form a dipeptide. After washing, the protecting group is removed and a third amino acid with an alpha protecting group is added to the dipeptide. This process is continued until a peptide of a desired length and sequence is obtained.
Other techniques have also been described. These methods include the synthesis of peptides on 96 plastic pins which fit the format of standard microtiter plates. Advanced techniques for synthesizing large numbers of molecules in an efficient manner have also been disclosed. Most notably, U.S. Pat. No. 5,143,854 (Pirrung et al.) and PCT Application No. 92/10092 disclose improved methods of molecular synthesis using light directed techniques. According to these methods, light is directed to selected regions of a substrate to remove protecting groups from the selected regions of the substrate. Thereafter, selected molecules are coupled to the substrate, followed by additional irradiation and coupling steps.
Methods, devices, and compositions for synthesis and use of diverse molecular sequences on a substrate are disclosed, as well as applications thereof.
A preferred embodiment of the invention provides for the synthesis of an array of polymers in which individual monomers in a lead polymer are systematically substituted with monomers from one or more basis sets of monomers. The method requires a limited number of masks and a limited number of processing steps. According to one specific aspect of the invention, a series of masking steps are conducted to first place the first monomer in the lead sequence on a substrate at a plurality of synthesis sites. The second monomer in the lead sequence is then added to the first monomer at a portion of the synthesis sites, while different monomers from a basis set are placed at discrete other synthesis sites. The process is repeated to produce all or a significant number of the mono substituted polymers based on the lead polymer using a given basis set of monomers. According to a preferred aspect of the invention, the technique uses light directed techniques, such as those described in Pirrung et al., U.S. Pat. No. 5,143,854.
Another aspect of the invention provides for efficient synthesis and screening of cyclic molecules. According to a preferred aspect of the invention, cyclic polymers are synthesized in an array in which the polymers are coupled to the substrate at different positions on the cyclic polymer ring. Therefore, a particular polymer may be presented in various xe2x80x9crotatedxe2x80x9d forms on the substrate for later screening. Again, the cyclic polymers are formed according to most preferred embodiments with the techniques of Pirrung et al.
The resulting substrates will have a variety of uses including, for example, screening polymers for biological activity. To screen for biological activity, the substrate is exposed to one or more receptors such as an antibody, oligonucleotide, whole cells, receptors on vesicles, lipids, or any one of a variety of other receptors. The receptors are preferably labeled with, for example, a fluorescent marker, a radioactive marker, or a labeled antibody reactive with the receptor. The location of the marker on the substrate is detected with, for example, photon detection or auto-radiographic techniques. Through knowledge of the sequence of the material at the location where binding is detected, it is possible to quickly determine which polymer(s) are complementary with the receptor. The technique can be used to screen large numbers of peptides or other polymers quickly and economically.
A further understanding of the nature and advantages of the inventions herein may be realized by reference to the remaining portions of the specification and the attached drawings.